Part:BBa_K4040019

CR3022 scFv Sequence and Features

Assembly Compatibility:

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COMPATIBLE WITH RFC[10]
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COMPATIBLE WITH RFC[12]
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COMPATIBLE WITH RFC[21]
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COMPATIBLE WITH RFC[23]
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INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 352
Illegal NgoMIV site found at 688
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INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 568

Usage and Biology

CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor-binding domain (RBD) on the spike protein that is able to cross-react with SARS-CoV-2. A single-chain variable fragment (scFv) is not actually a fragment of an antibody, but instead is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL or vice versa. This protein retains the specificity of the original immunoglobulin, despite the removal of the constant regions and the introduction of the linker. These molecules were created to facilitate phage display, where it is highly convenient to express the antigen-binding domain as a single peptide. As an alternative, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. ScFvs have many uses, e.g., flow cytometry, immunohistochemistry, and as antigen-binding domains of artificial T cell receptors (chimeric antigen receptor). CR3022 scFv is an scFv protein derived from the antibody CR3022.

Background and detail description

A Broad-spectrum neutralizing antibody

CR3022 was previously isolated from a SARS survivor and neutralizes SARS-CoV [1], CR3022 was recently found to also be a cross-reactive antibody that can bind to both SARS-CoV-2 and SARS-CoV [2]. Recent crystal structure demonstrated that CR3022 targets a highly conserved cryptic epitope on the receptor binding domain (RBD) of the S protein [3]. The CR3022 epitope is exposed only when the RBD is in the “up” but not the “down” conformation on the S protein. A few SARS-CoV-2 antibodies from COVID-19 patients have also recently been shown to target the CR3022 epitope, suggesting that it is an important site of vulnerability for the antibody response in SARS-CoV-2 infection. Out of 28 residues in the CR3022 epitope, 24 are conserved between SARS-CoV-2 and SARS-CoV, explaining the cross-reactive binding of CR3022. However, CR3022 has a higher affinity to SARS-CoV than to SARS-CoV-2 (>100-fold difference), and can neutralize SARS-CoV, but not SARS-CoV-2, in a live virus neutralization assay [3]. Therefore, CR3022 provides a good case study to probe antigenic variation between SARS-CoV-2 and SARS-CoV and the effects on antibody cross-neutralization.

Figure 1: Crystal structure of SARS-CoV receptor binding domain in complex with human antibody CR3022. The structure has been resolved [4] available in PDB with accession number 7JN5.

Binding to RBD on the spike protein and neutralizing

While it is now known that SARS-CoV and SARS-CoV-2 differ in antigenicity despite relatively high sequence conservation, there is a paucity of understanding of the underlying molecular determinants of these antigenic changes and the structural consequences of these differences. While CR3022 cannot neutralize SARS-CoV-2 WT in almost all studies, it can neutralize the SARS-CoV-2 P384A mutant. The KD of CR3022 Fab to SARS-CoV-2 WT RBD is 68 nM, whereas to SARS-CoV-2 P384A RBD is 1 nM, indicating that the affinity threshold for neutralization of SARS-CoV-2 to this epitope is in the low nM range. However, despite having a low nM affinity to SARS-CoV-2 P384A RBD, CR3022 only weakly neutralizes SARS-CoV-2 P384A with an IC50 of 3.2 μg/ml and SARS-CoV with an IC50 of 5.2 μg/ml.

Figure 2: Negative-stain EM and cryo-EM analysis of SARS spike bound to CR3022 Fab. TA) Representative 2D nsEM class averages of the trimeric SARS-CoV spike glycoprotein complexed with three CR3022 Fabs. (B) Side and top view of the 3D reconstruction showing CR3022 Fabs bound to all 3 RBDs on the SARS-CoV spike. The SARS-CoV RBD-CR3022 complex from the crystal structure is fitted into the nsEM density with the RBD shown in pink and CR3022 Fab in blue. (C) Side views of the B-factor-sharpened cryo-EM maps (transparent gray surface representation) representing three different classes of SARS spike with CR3022 Fab with different RBD-Fab orientations. While four different classes were identified, only three classes are shown here because classes 2 and 4 are very similar . The RBD-Fab complex model is fit into the densities with the RBDs shown in pink and CR3022 Fabs represented in blue. The atomic model of the apo SARS-CoV spike (PDB 6ACD) is also fit into density with one RBD removed for clarity. The protomers are colored in purple, magenta and deep magenta. (D) Top view of the class 2 cryo-EM map depicting potential quaternary contacts between the RBD-bound Fab and the spike NTD in this conformation. In this RBD-Fab conformation, the Fab would clash with the “down” RBD of the adjacent protomer (magenta) and, therefore, the adjacent RBD can only exist in an “up” conformation. (E) A close-up view of the Fab-spike interface showing the superimposition of CR3022 Fab and adjacent RBD. The residues that can contribute to quaternary interactions between CR3022 light chain and the NTD in two of the four classes (2 and 4) are shown. Date are from [4].

Used for a CAR

Chimeric antigen receptor T cells (also known as CAR T cells) are T cells that have been genetically engineered to produce an artificial T cell receptor for use in immunotherapy. Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are receptor proteins that have been engineered to give T cells the new ability to target a specific protein. The receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor. CAR-T cell therapy uses T cells engineered with CARs for cancer therapy. The premise of CAR-T immunotherapy is to modify T cells to recognize cancer cells in order to more effectively target and destroy them. Scientists harvest T cells from people, genetically alter them, then infuse the resulting CAR-T cells into patients to attack their tumors. CAR T cells can be both CD4+ and CD8+, with a 1-to-1 ratio of both cell types providing synergistic antitumor effects. CAR-T cells can be either derived from T cells in a patient's own blood (autologous) or derived from the T cells of another healthy donor (allogeneic). Once isolated from a person, these T cells are genetically engineered to express a specific CAR, which programs them to target an antigen that is present on the surface of tumors. For safety, CAR-T cells are engineered to be specific to an antigen expressed on a tumor that is not expressed on healthy cells. After CAR-T cells are infused into a patient, they act as a "living drug" against cancer cells. When they come in contact with their targeted antigen on a cell, CAR-T cells bind to it and become activated, then proceed to proliferate and become cytotoxic.CAR-T cells destroy cells through several mechanisms, including extensive stimulated cell proliferation, increasing the degree to which they are toxic to other living cells (cytotoxicity), and by causing the increased secretion of factors that can affect other cells such as cytokines, interleukins, and growth factors. The first CAR-T cell therapies were FDA-approved in 2017, and there are now 5 approved CAR-T therapies.

Figure 3: The diagram above represents the process of chimeric antigen receptor T cell therapy (CAR), this is a method of immunotherapy, which is a growing practice in the treatment of cancer. The final result should be a production of equipped T cells that can recognize and fight the infected cancer cells in the body. 1. T cells (represented by objects labeled as ’t’) are removed from the patient's blood. 2. Then in a lab setting the gene that encodes for the specific antigen receptors is incorporated into the T cells. 3. Thus producing the CAR receptors (labeled as c) on the surface of the cells. 4. The newly modified T cells are then further harvested and grown in the lab. 5. After a certain time period, the engineered T cells are infused back into the patient.(Figure from wikipedia)

Chimeric antigen receptors combine many facets of normal T cell activation into a single protein. They link an extracellular antigen recognition domain to an intracellular signaling domain, which activates the T cell when an antigen is bound. CARs are composed of four regions: an antigen recognition domain, an extracellular hinge region, a transmembrane domain, and an intracellular T cell signaling domain.

Antigen recognition domain

The antigen recognition domain is exposed to the outside of the cell, in the ectodomain portion of the receptor. It interacts with potential target molecules and is responsible for targeting the CAR-T cell to any cell expressing a matching molecule.

Figure 4: Different components of a chimeric antigen receptor.(Figure from wikipedia)

The antigen recognition domain is typically derived from the variable regions of a monoclonal antibody linked together as a single-chain variable fragment (scFv). An scFv is a chimeric protein made up of the light (VL) and heavy (VH) chains of immunoglobins, connected with a short linker peptide. These VL and VH regions are selected in advance for their binding ability to the target antigen (such as CD19). The linker between the two chains consists of hydrophilic residues with stretches of glycine and serine in them for flexibility as well as stretches of glutamate and lysine for added solubility.Single domain antibodies (e.g. VH, VHH) have been engineered and developed as antigen recognition domains in the CAR format due to their high transduction efficiency in T cells. In addition to antibody fragments, non‐antibody‐based approaches have also been used to direct CAR specificity, usually taking advantage of ligand/receptor pairs that normally bind to each other. Cytokines, innate immune receptors, TNF receptors, growth factors, and structural proteins have all been successfully used as CAR antigen recognition domains.

When CR3022 scFv is used for a CAR, the CAR receptor can recognize the SARS-COV and SARS-COV-2 RBD.

Experimental results

In our project, to program engulfment based on recognition of the SARS-CoV-2 spike protein, we used a CAR design for the synthetic receptor strategy in our study. The synthetic receptors were constructed to contain an scFv derived from CR3022, and the CD8 transmembrane domain present in the αCD19 CAR for T cells . For the cytoplasmic domains, we used the common γ subunit of Fc receptors (CARγ), MEGF10 (CARMEGF10), MERTK (CARMERTK) and CD3ζ (CARζ) in our study (Figure 5A). These cytoplasmic domains are capable of promoting phagocytosis by macrophages.

Figure 5: CARs mediate phagocytosis of SARS-CoV-2 virions. (A) Different cell lines were inoculated with a SARS-CoV-2 pseudotyped virus. At 16 h post inoculation, pseudotyped virus entry was analyzed by determining the luciferase activity in cell lysates. Signals obtained for particles bearing no envelope protein were used for normalization. The average of three independent experiments is shown. Error bars indicate the SEM. (B) The uptake of pseudotyped virions by UTD and CAR macrophages was analyzed by flow cytometry. Different cell lines were stained with an anti-S primary Ab. The histograms shown in black correspond to the isotype controls, whereas the red histograms indicate positive fluorescence. Data are reported as the phagocytic score (% positive cells x MFI, right panel). (C) Cell lines were infected with the SARS-CoV-2 pseudotyped virus or mock infected. Cytokine levels in the supernatants were determined by a multiplex bead array. The relative level was calculated as the ratio of the infected cells to the mock-infected THP-1 cells. Data are shown as the mean ± s.d.

Next, we used lentiviral vector technology to express the fusion constructs in human macrophage THP-1 cells using clinically validated techniques. The cDNA sequences containing the various fusion constructs were cloned into a third-generation lentiviral vector in which the CMV promoter was replaced with the EF-1α promoter. An extracellular MYC epitope was cloned into the receptors to permit detection by flow cytometry. Lentiviral vector supernatants transduced THP-1 cells with high efficiency (Figure 5B). The phagocytic potential of human macrophage THP-1 cell lines expressing different CAR receptors or a truncated CAR receptor (CARΔ) lacking the intracellular domain was measured with a cell-based assay.CAR macrophages and control untransduced (UTD) macrophages did not show notable phagocytosis of 293 cells; however, CARMEGF10, CARγ and CARζ cells but not CARMERTK, CARΔ, or UTD macrophages phagocytosed Spike-bearing 293 cells in an S-specific manner (Figure 5C).

References

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